Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
J Biol Chem. 2010 May 7;285(19):14415-23. doi: 10.1074/jbc.M109.074583. Epub 2010 Mar 12.
PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins.
PIASy 是一种小泛素相关修饰酶（SUMO）连接酶，可修饰有丝分裂非洲爪蟾卵提取物中的染色体蛋白，并在有丝分裂染色体分离中发挥重要作用。我们分离到一种新型 SUMO-2/3 修饰的有丝分裂染色体蛋白，并将其鉴定为多聚（ADP-核糖）聚合酶 1（PARP1）。PARP1 在有丝分裂染色体上与 SUMO-2/3 强烈结合，但不在间期间质染色质上结合。PIASy 可促进卵提取物和体外重新构成 SUMO 化测定中 PARP1 的 SUMO 化。通过对有丝分裂中 SUMO 化的 PARP1 的串联质谱分析，我们鉴定出 PARP1 的 BRCA1 C 端结构域内的一个残基（赖氨酸 482）是其主要 SUMO 化位点。该残基的突变显著降低了卵提取物中 PARP1 的 SUMO 化，并增强了使用纯化成分进行的测定中来自修饰次要赖氨酸残基的物质的积累。SUMO 化的 PARP1 不会改变体外 PARP1 酶活性、聚 ADP-核糖化（PAR 化），也不会抑制 PARP1 的 SUMO 化改变 PARP1 在有丝分裂染色体上的积累，这表明 SUMO 化既不调节 PARP1 的内在活性，也不调节其定位。然而，SUMO 化的缺失增加了 PARP1 对分离染色体上的 PAR 化的依赖性，表明 SUMO 化控制 PARP1 修饰其他染色质相关蛋白的能力。
